Effects of freezing storage time on the density of muscarinic receptors in the human postmortem brain: an autoradiographic study in control and Alzheimer's disease brain tissues.
Author(s): Rodŕiguez-Puertas R, Pascual J, Pazos A
Publication: Brain Res, 1996, Vol. 728, Page 65-71
PubMed ID: 8864298 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if the duration of frozen storage (-25°C) affects the density of muscarinic receptors (MR) detected by a radioligand binding assay in snap-frozen brain specimens from postmortem patients with Alzheimer’s disease (AD) and those without a neuropsychiatric disorder; the potential influence of postmortem interval (PMI), patient age, and patient gender on MR density was also investigated.
Conclusion of Paper
In brain specimens from donors without a neurological or psychiatric disorder (controls), the density of MR was not significantly affected by donor age or PMI and was comparable between specimens from male and female donors. MR density was significantly reduced when storage at -25°C exceeded 39 months in specimens from the frontal cortex (-7.6% to -13.3% per year in layers I-VI; P<0.0001), hippocampus (-6.3% to -8.8% per year in the pyramidal layers; P<0.01), and caudate nucleus (-8.0% per year; P<0.005), but not in the putamen or hippocampal dentate gyrus.
In brain specimens from AD donors, MR density declined significantly with donor age in frontal cortex layers I-III (P<0.025), IV (P<0.025), and V-VI (P<0.01) but not in specimens from the entorhinal cortex, hippocampus, or striatum. MR density did not differ between specimens from male and female donors diagnosed with AD. MR density decreased significantly with storage at -25°C in specimens from the frontal cortex (declines ranged from -12.3% to -14.2% per year in layers I-VI; P<0.0001), in the entorhinal cortex (-12.3% per year; P≤0.01), and the pyramidal layer of CA1 (-12.3% per year; P≤0.05) but not in the CA3 field, the hippocampus, caudate nucleus, putamen nucleus, or hippocampal dentate gyrus.
When AD and control donors were matched for age, gender, PMI, and frozen storage duration, significant differences in MR density were limited to a reduction (-30%) in the hilus of the dentate gyrus of AD donors (P<0.022).
Studies
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Study Purpose
The purpose of this study was to determine if the duration of frozen storage (-25°C) affects the density of muscarinic receptors (MR) detected by a radioligand binding assay in snap-frozen brain specimens from postmortem patients with Alzheimer’s disease and those without a neuropsychiatric disorder; the potential influence of postmortem interval (PMI), patient age, and patient gender on MR density was also investigated. Brains specimens from 41 individuals (25 males, 16 females; 41-84 y; PMI range: 1-71 h) without a history of a neurological or psychiatric disorder and from 18 individuals diagnosed with Alzheimer’s disease (AD) (6 males, 12 females; 51-83 y; PMI range: 2-48 h) were collected postmortem and divided sagitally. A diagnosis of Alzheimer’s disease was based on staining of multiple samples using the “Cross’ modification of the Palmgren’s silver technique”. Brain regions from one hemibrain were dissected and frontal cortex, hippocampus, and striatum specimens were snap-frozen on dry ice, stored at -80°C for ≥48 h, and then transferred to -25°C for long-term storage (control specimens: 8-75 months; AD specimens: 12-78 months). Frozen sections (10 µm thick) were cut and mounted on gelatin-coated slides 1 week before analysis and stored at -25°C. MR were labeled by radioligand binding assay using 1.5 nM [3H]N-methylscopolamein ([3H]NMS) and non-specific binding was determined with 10 µM atropine.
Summary of Findings:
In brain specimens from donors without a neurological or psychiatric disorder (controls), the density of MR was not significantly affected by donor age or PMI and was comparable between specimens from male and female donors. MR density was significantly reduced when storage at --25°C) exceeded 39 months in specimens from frontal cortex (declines ranged from -7.6% to -13.3% per year in layers I-VI; P<0.0001), in hippocampus (declines ranged from -6.3% to -8.8% per year in the pyramidal layers; P<0.01), and caudate nucleus (-8.0% per year; P<0.005), but not in the putamen or hippocampal dentate gyrus.
In brain specimens from AD donors, MR density declined significantly with donor age in frontal cortex layers I-III (P<0.025), IV (P<0.025), and V-VI (P<0.01) but not in specimens from the entorhinal cortex, hippocampus, or striatum. MR density did not differ between specimens from male and female donors diagnosed with AD. MR density decreased significantly with storage at -25°C) in specimens from frontal cortex (declines ranged from -12.3% to -14.2% per year in layers I-VI; P<0.0001), in the entorhinal cortex (-12.3% per year; P≤0.01), and the pyramidal layer of CA1 (-12.3% per year; P≤0.05) but not in the CA3 field, the hippocampus, caudate nucleus, putamen nucleus, or hippocampal dentate gyrus.
The mean duration of frozen storage differed significantly between control and AD cohorts (40 versus 58 months, respectively; P≤0.005). When AD and control donors were matched for age, gender, PMI, and frozen storage duration, significant differences in MR density were limited to a reduction (-30%) in the hilus of the dentate gyrus of AD donors (P<0.022).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Alzheimer's Disease
- Autopsy
- Normal
Platform:
Analyte Technology Platform Protein Receptor binding Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient age 41-84 y (normal control specimens)
51-83 y (Alzheimer’s disease specimens)
Preaquisition Patient gender Female
Male
Preaquisition Postmortem interval 1-71 h (normal control specimens)
2-48 h (Alzheimer’s disease specimens)
Storage Storage duration 8-75 months at -25°C (normal control specimens)
12-78 months at -25°C (Alzheimer’s disease specimens)
Biospecimen Acquisition Biospecimen location Frontal cortex
Hippocampus
Striatum